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miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling

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RNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2298))

Abstract

The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.

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Acknowledgments

This work was supported by UCSF Program for Breakthrough Biomedical Research (PBBR) Postdoctoral Grant (to V.S.), NIAID R01AI137270 (to D.G.F.), UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation (to D.E.W.), and NIH Director’s Early Independence Award DP5OD017895 (to D.E.W.).

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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

    Vanja Stojković, David E. Weinberg & Danica Galonić Fujimori

  2. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA

    Danica Galonić Fujimori

Authors
  1. Vanja Stojković
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  2. David E. Weinberg
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  3. Danica Galonić Fujimori
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Corresponding author

Correspondence to Danica Galonić Fujimori .

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Editors and Affiliations

  1. Department of Urology, University of California, San Francisco, San Francisco, CA, USA

    Mary McMahon

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Stojković, V., Weinberg, D.E., Fujimori, D.G. (2021). miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling. In: McMahon, M. (eds) RNA Modifications. Methods in Molecular Biology, vol 2298. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1374-0_7

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  • DOI: https://doi.org/10.1007/978-1-0716-1374-0_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1373-3

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